Project Summary/Abstract The scale of the US opioid addiction crisis clearly evidences the critical need for breakthroughs to assess compound abuse liabilities and to enable discovery of new analgesics with minimal abuse potential. We hypothesize that altered addiction-associated physiology can be modeled in co-cultured hiPSC-derived neuronal and astrocyte cells and detected by profiling gene expression alterations and associated phenotypic readouts. Our proposal addresses the significant lack of physiologically-relevant tools capable of profiling relevant molecular changes that underpin addiction in the human nervous system by executing Specific Aims that encompass: (1) generation of a panel of multicolor hiPSC-derived neuronal and astrocyte cells with stable lineage specific fluorescent reporters; (2) execution of a phenotype-based pilot machine learning-enabled predictive abuse liability screen; and (3) combination of phenotypic profiling and gene expression analysis to characterize the molecular and cellular changes associated with compound exposure. Successful completion of our project will deliver 2 significant advances: (1) an important new technical framework to assess abuse potential of candidate therapeutics early in the development process and (2) an innovative, scalable in vitro assay platform that enables discovery of targets and associated candidate therapeutics to mitigate addiction phenotypes. These innovations will position Cairn Biosciences to initiate drug discovery programs that will bring significant societal benefit by helping to stem the growth of the US opioid crisis and reduce the mortality rates and economic burden associated with this National Emergency.